The research proposed in this grant application encompasses two main areas: l. The study of the mechanism of action of putrescine oxidase (a simple example of flavoprotein oxidases). The stoichiometry and mechanism of the oxidation of mercaptans by the enzyme and the inhibition of this oxidation by cyanide will be determined with emphasis on the hypothesis that these substrates may form a covalent intermediate with the (single) FAD during catalysis. Flavin binding to the apoenzyme will be studied using circular dichroism, difference spectroscopy and potential flavin binding inhibitors. Further mapping of the active site will be attempted using dye-sensitized photoinactivation as a probe, and the kinetics of the overall oxidation of putrescine will be determined using the rapid-scan stopped-flow apparatus developed in the laboratory. 2. The second area of proposed research is the application of on-line computer techniques to CD spectroscopy and to a stopped-flow CD system. In addition, a 100 KHZ dual-wavelength optical system will be developed for use with rapid reaction techniques such as T-jump, flash photolysis and stopped-flow.